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Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

Kanevsky M, Aldrich RW - J. Gen. Physiol. (1999)

Bottom Line: We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion.Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating.These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

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Mutations of F401 and cooperative gating: sigmoidicity. (A) Activation families of wt currents taken at voltages between −65 and 25 mV, F401L (−80 to +30 mV), and F401A (-70 mV to +90 mV) in 10-mV increments. (B) Data transformed to normalize the current amplitude and overall speed of activation for the voltages shown. Sigmoidicity is defined as the amount of initial delay relative to the overall rate of activation. Its voltage dependence can be visualized by following transformation (Zagotta et al. 1994a). The currents from different voltage steps are first scaled to match at their peaks. Then the time derivative of the current waveform is determined at the time when current amplitude is half maximal. In general, it is at that point that the slope is the steepest. All the records in the family are then expanded or compressed along the time axis so that their derivatives at half-maximum match. This transformation results in parallel curves whose relative displacement along the x axis is the measure of sigmoidicity. The noisier traces correspond to lower voltages. Data were filtered at between 8 and 10 kHz and sampled every 20–50 μs.
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Figure 10: Mutations of F401 and cooperative gating: sigmoidicity. (A) Activation families of wt currents taken at voltages between −65 and 25 mV, F401L (−80 to +30 mV), and F401A (-70 mV to +90 mV) in 10-mV increments. (B) Data transformed to normalize the current amplitude and overall speed of activation for the voltages shown. Sigmoidicity is defined as the amount of initial delay relative to the overall rate of activation. Its voltage dependence can be visualized by following transformation (Zagotta et al. 1994a). The currents from different voltage steps are first scaled to match at their peaks. Then the time derivative of the current waveform is determined at the time when current amplitude is half maximal. In general, it is at that point that the slope is the steepest. All the records in the family are then expanded or compressed along the time axis so that their derivatives at half-maximum match. This transformation results in parallel curves whose relative displacement along the x axis is the measure of sigmoidicity. The noisier traces correspond to lower voltages. Data were filtered at between 8 and 10 kHz and sampled every 20–50 μs.

Mentions: Shaker ionic currents activate upon depolarization with a delay, giving rise to a sigmoidal time course. This sigmoidicity arises from the multi-step nature of the activation process. Voltage dependence in the sigmoidicity of ionic currents is a diagnostic feature of deviation from subunit independence in activation (Zagotta et al. 1994a; Smith-Maxwell et al. 1998a,Smith-Maxwell et al. 1998b). Sigmoidicity is preserved in the mutant channels F401L and F401A (Fig. 10 A), although their overall kinetics and the absolute amount of delay vary. For a noncooperative model of channel activation that postulates n independent first-order voltage-dependent processes (Hodgkin and Huxley 1952; Cole and Moore 1960), it can be shown that sigmoidicity will be the same for a given n at all test voltages, and the time- and amplitude-scaled traces will superimpose. Over the voltage ranges shown, wt and F401L channels clearly display deviations from the independent scheme. Sigmoidicity is greater at higher than at lower voltages, but appears to reach a saturating value when the voltage is in the range of maximal steady state activation (reached near or below 0 mV for both of these channels). Zagotta et al. 1994a used this observation in wt Shaker to argue for a form of cooperativity that acts to slow the first closing transition from the open state causing the current waveform at lower voltages to be close to a monoexponential function because it is limited by the slow final step (see also Smith-Maxwell et al. 1998a,Smith-Maxwell et al. 1998b; Ledwell and Aldrich 1999). From the F401L records, it is apparent that the smaller amount of sigmoidicity at the lower voltages is at least as pronounced as in the wt (note that the steady state activation in F401L is shifted negatively relative to the wt by 15–20 mV). On the other hand, over the voltage range between −40 and +90 mV, channels with an alanine substitution at F401 do not display the clear increase in the amount of sigmoidicity with higher depolarizations seen with the wt and F401L. The F401A mutant acts as though there is no rate-limiting transition present at the lower voltages, implying that the leaving rate from the open state is not slowed relative to the predictions of a mechanism with independently gating subunits.


Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

Kanevsky M, Aldrich RW - J. Gen. Physiol. (1999)

Mutations of F401 and cooperative gating: sigmoidicity. (A) Activation families of wt currents taken at voltages between −65 and 25 mV, F401L (−80 to +30 mV), and F401A (-70 mV to +90 mV) in 10-mV increments. (B) Data transformed to normalize the current amplitude and overall speed of activation for the voltages shown. Sigmoidicity is defined as the amount of initial delay relative to the overall rate of activation. Its voltage dependence can be visualized by following transformation (Zagotta et al. 1994a). The currents from different voltage steps are first scaled to match at their peaks. Then the time derivative of the current waveform is determined at the time when current amplitude is half maximal. In general, it is at that point that the slope is the steepest. All the records in the family are then expanded or compressed along the time axis so that their derivatives at half-maximum match. This transformation results in parallel curves whose relative displacement along the x axis is the measure of sigmoidicity. The noisier traces correspond to lower voltages. Data were filtered at between 8 and 10 kHz and sampled every 20–50 μs.
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Related In: Results  -  Collection

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Figure 10: Mutations of F401 and cooperative gating: sigmoidicity. (A) Activation families of wt currents taken at voltages between −65 and 25 mV, F401L (−80 to +30 mV), and F401A (-70 mV to +90 mV) in 10-mV increments. (B) Data transformed to normalize the current amplitude and overall speed of activation for the voltages shown. Sigmoidicity is defined as the amount of initial delay relative to the overall rate of activation. Its voltage dependence can be visualized by following transformation (Zagotta et al. 1994a). The currents from different voltage steps are first scaled to match at their peaks. Then the time derivative of the current waveform is determined at the time when current amplitude is half maximal. In general, it is at that point that the slope is the steepest. All the records in the family are then expanded or compressed along the time axis so that their derivatives at half-maximum match. This transformation results in parallel curves whose relative displacement along the x axis is the measure of sigmoidicity. The noisier traces correspond to lower voltages. Data were filtered at between 8 and 10 kHz and sampled every 20–50 μs.
Mentions: Shaker ionic currents activate upon depolarization with a delay, giving rise to a sigmoidal time course. This sigmoidicity arises from the multi-step nature of the activation process. Voltage dependence in the sigmoidicity of ionic currents is a diagnostic feature of deviation from subunit independence in activation (Zagotta et al. 1994a; Smith-Maxwell et al. 1998a,Smith-Maxwell et al. 1998b). Sigmoidicity is preserved in the mutant channels F401L and F401A (Fig. 10 A), although their overall kinetics and the absolute amount of delay vary. For a noncooperative model of channel activation that postulates n independent first-order voltage-dependent processes (Hodgkin and Huxley 1952; Cole and Moore 1960), it can be shown that sigmoidicity will be the same for a given n at all test voltages, and the time- and amplitude-scaled traces will superimpose. Over the voltage ranges shown, wt and F401L channels clearly display deviations from the independent scheme. Sigmoidicity is greater at higher than at lower voltages, but appears to reach a saturating value when the voltage is in the range of maximal steady state activation (reached near or below 0 mV for both of these channels). Zagotta et al. 1994a used this observation in wt Shaker to argue for a form of cooperativity that acts to slow the first closing transition from the open state causing the current waveform at lower voltages to be close to a monoexponential function because it is limited by the slow final step (see also Smith-Maxwell et al. 1998a,Smith-Maxwell et al. 1998b; Ledwell and Aldrich 1999). From the F401L records, it is apparent that the smaller amount of sigmoidicity at the lower voltages is at least as pronounced as in the wt (note that the steady state activation in F401L is shifted negatively relative to the wt by 15–20 mV). On the other hand, over the voltage range between −40 and +90 mV, channels with an alanine substitution at F401 do not display the clear increase in the amount of sigmoidicity with higher depolarizations seen with the wt and F401L. The F401A mutant acts as though there is no rate-limiting transition present at the lower voltages, implying that the leaving rate from the open state is not slowed relative to the predictions of a mechanism with independently gating subunits.

Bottom Line: We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion.Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating.These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

Show MeSH
Related in: MedlinePlus